Liquid chromatography is used to separate the components of a sample substance by passing an eluent containing the sample through a column. The components of the sample in the eluent stream have different retention times within the column, and therefore exit the column in a particular sequence depending on the nature of the components of the sample and the nature of the column. The sequence of components is detected, for example, photometrically by measuring the intensity of light absorbed by the eluent stream.
Developments in liquid chromatography have led beyond component separation and analysis to ion separation and analysis. Ion exchange liquid chromatography is often complicated by limitations in the capability to detect the eluted sample ions in the column effluent. For example, many inorganic and organic ions are non light-absorbing and difficult to detect using conventional photometric detectors. Even though the separation of such "transparent" ions may be conveniently effected using ion exchange resin columns, the detection and measurement of these transparent ions by conventional photometric means is ineffective since they are optically indistinguishable from the transparent eluents commonly prescribed by the art. Hence, practices using photometers to detect ions in effluent have been effective only when the ions to be analyzed either contain chromophores or can generate chromophores through post-column reactions with appropriate reagents.
The problems of transparent ion detection are in part solved by the recent development of Indirect Photometric Chromatography (IPC). IPC has been described in detail in U.S. Pat. No. 4,414,842. IPC involves a method and apparatus for measuring ions in a sample undergoing chromatographic analysis wherein the ions of interest are transparent at the wavelengths monitored by the photometer. In the IPC method, these ions are first displaced from the ion exchange column in which they have been selectively adsorbed, by passing through the column, an eluent containing eluting ions which are or are made light absorbing. The displaced sample ions as they appear in the eluent are then detected in series and quantified by observing the decrements the ions cause in eluent absorbance, as revealed by photometric monitoring. For further details of IPC principles and techniques, see, "Indirect Photometric Chromatography", Anal. Chem., 1982, 54, 462-469, written by Small and Miller. Both of the above references are incorporated herein.
A problem with the known IPC method arises when an attempt is made to carry out independent analysis of both anions and cations, both of which are transparent. Since such ions in the mobile or eluent phase both contribute to the absorbance of UV wavelengths, it has not heretofore been found possible to measure independently, the eluent absorbance due to the individual anions and the individual cations.
One attempt was made to detect and record anions and cations eluting simultaneously from a column; see Yamamoto, Yamamoto, Yamamoto, Matsushita, Baba and Ikushige, "Simultaneous Determination of Inorganic Anions and Cations by Ion Chromatography with Ethylenediaminetetraacetic Acid as Eluent" Anal. Chem., 56, 832-834, (1984). This attempt was not successful in independently determining the anion and cation concentrations as they eluted. The scientists noted that, "The retention times observed for Ca.sup.2+ and Mg.sup.2+, injected as metal cations, and those injected as EDTA chelate anions were not significantly different." To overcome this problem, the scientists converted the Mg.sup.2+ and Ca.sup.2+ cations to chelate anions using the EDTA eluent, separated the anion species for detection, and detected the anions using conventional chromatographic methods. The scientists specifically noted that Mg.sup.2+ and Ca.sup.2+ could not be found except as corresponding anion peaks in the chromatogram.
Even though detection systems have been developed for differentiating among pure components of an eluent stream, e.g., see U.S. Pat. No. 4,367,041, no detection system presently exists which can distinguish between transparent anion and cation species having identical or nearly identical retention times.
The present invention has as its object to provide a method and apparatus using indirect photometric chromatography techniques to detect independently anion and cation species of a sample within a single chromatograph even when the species have identical or nearly identical retention times. The technique of the present invention displaces sample anions and cations with photometrically monitorable eluent anion and cation species. The present invention teaches the use of an eluent containing a salt with chromophoric anion and cation species each having a known characteristic ratio of absorbance at predetermined wavelengths, and the use of certain mathematical equations to develop, independently two chromatograms, one for anions, and one for cations.
Other objects, features and advantages of the present invention will become apparent upon reading the following detailed description and examples, when taken together with the drawing and appended claims.